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Home , Microinjection, Transduction (genetics), Transfection

ibidi Application Guide

The Principle of Transfection . . . . . 2

Transfection Methods ...... 3

Chemical Transfection ...... 3

Lipofection ...... 3 Membrane Fusion ...... 4 Membrane Fusion vs . Lipofection ...... 4 Calcium Phosphate ...... 5 Cationic Polymers ...... 5

Viral ...... 6

Adenoviral Transduction ...... 6 Lentiviral Transduction ...... 7 Method Comparison ...... 7 Selected Publications

Physical Transfection ...... 8 F. Martin-Sanchez et al. Inflammasome-dependent IL-1[beta] release depends upon membrane permeabilisation. Death ...... 8 Differ, 2016, 10.1038/cdd.2015.176

Electroporation ...... 8 M. Moch et al. Effects of Plectin Depletion on Keratin Network Dynamics and Organization. PloS one, 2016, 10.1371/journal. Experimental Example ...... 9 pone.0149106 G. Burgstaller, S. Vierkotten, M. Lindner, M. Königshoff and O. Eickelberg. Multidimensional immunolabeling and 4D time- lapse imaging of vital ex vivo lung tissue. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2015, 10.1152/ajplung.00061.2015

A. Csiszar, N. Hersch, S. Dieluweit, R. Biehl, R. Merkel and B. Hoffmann. Novel fusogenic for fluorescent cell labeling and membrane modification. Bioconjugate chemistry, 2010, 10.1021/bc900470y

C. Kleusch, N. Hersch, B. Hoffmann, R. Merkel and A. Csiszár. Fluorescent : Functional parts of fusogenic liposomes and tools for labeling and visualization. Molecules, 2012, 10.3390/molecules17011055

.com The Principle of Transfection

siRNA

Fuse-It-siRNA mRNA Particle Fuse-It-mRNA Adenoviral Transduction CAR

Membrane Fusion Endosome

ds-siRNA ssRNA RELEASE OF RNA PROTEIN REVERSE Protein EXPRESSION ss-siRNA

DEGRADATION RELEASE dsDNA OF DNA TRANSLATION mRNA Lentiviral RISC mRNA Transduction Virus Particle

Protein

STABLE TRANSCRIPTION INTEGRATION

TRANSLATION mRNA

LYSOSOMAL DEGRADATION Microinjection

Lipofection RELEASE OF DNA Plasmid DNA DNA Lysosome

Transfection – Reagent – + Endosome – + + Plasmid DNA

Electroporation Plasmid DNA Ca2+ Ca2+

Ca2+ Ca2+ Ca2+ Ca2+ Calcium Ca2+ Phospate Ca2+ Ca2+ + + Plasmid + + DNA Calcium + + + Phospate + + + Cationic Cationic + Polymers Polymers Transfection is defined as the process of inserting nucleic acids (e g. ,. plasmid DNA, cDNA, mRNA, miRNA, siRNA) into the cytoplasm of eukaryotic cells . In addition, and such as beads or dyes can be transfected . In the field of cell , transfection is an important and widely used tool for analyzing the function of various genes . This method can be used for gene silencing via RNAi, gene editing via CRISPR/Cas9, as well as overexpression studies by cellular integration of plasmid DNA, mRNA, or proteins . Many researchers conduct transfection experiments on a daily basis, which requires a concise understanding of the biological background, precise and detailed planning, and a robust transfection protocol .

Kim T.K., Eberwine J.H. Mammalian cell transfection: The present and the future. Anal Bioanal Chem, 2010, 10.1007/ s00216-010-3821-6

2 Transfection Methods

Naturally, cells usually do not take up foreign nucleic acids without external stimulation . Thus, an efficient and biocompatible transfection method is needed, which has to be accurately adapted to the cell type and the material to be transferred . Many transfection methods have been developed, which can be basically subdivided into three different groups: chemical transfection, physical transfection, and viral transduction . In this Application Guide, you can find a brief description of the most commonly applied methods, summarizing the characteristics, advantages, and pitfalls of each approach .

Chemical Transfection Viral Transduction Physical Transfection

• Lipofection • Adenoviral Transduction • Microinjection • Membrane Fusion • Lentiviral Transduction • Electroporation • Calcium Phosphate • Cationic Polymers

Chemical Transfection

Lipofection

Protein

Applications: TRANSCRIPTION TRANSLATION mRNA

Lipofection is commonly used to transfer nucleic LYSOSOMAL acids such as RNA or DNA into eukaryotic cells . After DEGRADATION Lipofection RELEASE protocol optimization, this method is easy to apply OF DNA and yields highly reproducible results of transient Plasmid DNA Lysosome and stable . However, lipofection Transfection efficiency strongly depends on the cell type and has Reagent to be tested and optimized in advance . Especially Endosome for primary and non-dividing cells, the viability after the transfection process might be decreased due to the high cellular sensitivity . In contrast to membrane fusion, lipofection is based on endosomal molecule uptake, which might delay the time of analysis . ibidi Solutions: Principle: The ibidi µ-Slides, µ-Dishes, and µ-Plates are ideally suited for lipofection using various Usually, a mixture of neutral and cationic liposomes cell numbers and volumes . Volume calculations forms complexes with the nucleotides of interest . for frequently used sizes are available in the These complexes pass the cell membrane and instructions of each Torpedo reagent . then release the nucleotides into the cytoplasm via . Then, the nucleotides either successfully The pCMV/pCAG-LifeAct plasmid can be used escape the endosome or undergo lysosomal for non-toxic F- visualization in living cells . degradation, of which the latter might lead to reduced transfection efficiency . After endosomal escape, the nucleotide is expressed in the target cells .

Felgner P.L., et al. Lipofection: a highly efficient, -mediated DNA-transfection procedure. 1987, Proc Natl Acad Sci U S A, 10.1073/pnas.84.21.7413

3 Membrane Fusion Fusogenic Applications: Membrane fusion is a novel and highly superior transfection method to incorporate various molecules and particles into mammalian cells . Not only cells that undergo , but also primary and non-dividing Liposomal Carrier cells can be transfected by using this innovative method . It allows for highly efficient transfer of mRNA, siRNA, proteins, beads, dyes, and other molecules . Since the reagents are non-toxic to sensitive and FUSION difficult-to-transfect cells, even primary neurons, keratinocytes, and stem cells retain high viability after the transfection . Additionally, the very short incubation times minimize cell stress and provide an almost unaffected cell behavior after the molecule transfer .

Principle: IMMEDIATE RELEASE INTO THE CYTOPLASM Liposomal carriers, which consist of neutral and cationic lipids, are used to transfer the molecule of interest via membrane fusion into the cell . The molecules are first incorporated into the liposomal carriers, which upon contact, instantly fuse with the cell membrane . The included molecules are then directly released into the ibidi Solutions: cytoplasm without processes such as endocytosis and lysosomal degradation . This results in fusion The ibidi µ-Slides, µ-Dishes, and µ-Plates are efficiencies up to 80–100% within only seconds to a ideally suited for membrane fusion using various few minutes, depending on the cell type . cell numbers and volumes . Volume calculations for frequently used sizes are available in the Csiszár, A., et al. Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification. Bioconjug Chem, instructions of each Fuse-It reagent . 2010, 10.1021/bc900470y

Membrane Fusion vs . Lipofection

Membrane Fusion Is FusogenicFusogenic EndocytoticEndocytotic Superior to Lipofection LiposomeLiposome LiposomeLiposome

In contrast to lipofection,

membrane fusion does not LiposomalLiposomal LipoplexLipoplex involve endocytosis . Instead, CarrierCarrier the fusogenic liposomal carrier MembraneMembrane Fusion Fusion LipofectionLipofection simply fuses with the cell FUSIONFUSION membrane, then releases the included molecule of interest ENDOCYTOSISENDOCYTOSIS directly into the cytoplasm . This results in immediate IMMEDIATEIMMEDIATE RELEASE RELEASE and efficient transfer without INTOINTO THE THE CYTOPLASM CYTOPLASM lysosomal degradation .

EndosomeEndosome

ENDOSOMALENDOSOMAL RELEASERELEASE

LysosomeLysosome

LYSOSOMALLYSOSOMAL DEGRADATIONDEGRADATION

4 Calcium Phosphate

Applications: Protein TRANSCRIPTION

The calcium phosphate transfection is an inexpensive TRANSLATION mRNA and simple method for transient or stable nucleotide LYSOSOMAL DEGRADATION transfer into most cell lines .The transfection efficiency, RELEASE however, strongly depends on the cell constitution, Calcium OF DNA Phospate the pH, and the quality and the amount of the used Lysosome nucleotides . Therefore, the optimal experimental Plasmid conditions have to be tediously established in ad- DNA Ca2+ Ca2+ Ca2+ Ca2+ vance . Further, the calcium phosphate transfection Ca2+ Ca2+ Endosome Calcium Ca2+ is toxic and therefore not suitable for most sensitive Phospate Ca2+ primary cell lines . Ca2+

Principle: A mixture of the nucleotides, calcium, and phosphate buffer forms a precipitate that is taken up by the cells ibidi Solutions: via endocytosis . Then, the nucleotides either escape the endosome or undergo lysosomal degradation, of The ibidi µ-Slides, µ-Dishes, and µ-Plates are which the latter might lead to reduced transfection ideally suited for calcium phosphate transfection efficiency . Successfully escaped nucleotides can with various cell numbers . then be expressed in the target cells .

Cationic Polymers

Applications: Protein TRANSCRIPTION

Using cationic polymers, nucleotides can be tran- TRANSLATION mRNA siently transfected into eukaryotic cells in an LYSOSOMAL inexpensive and simple manner . After protocol opti- Cationic DEGRADATION Polymers RELEASE mization, the results can be easily reproduced . As OF DNA a drawback, transfection by cationic polymers has Plasmid DNA Lysosome a very low efficiency (<10%) in a number of cell + + types, including primary cells . Further, cationic poly- Cationic + + Polymers + + + + + + mers are highly cytotoxic and therefore not suitable + Endosome for transfection of sensitive cells and generation of stable cell lines .

Principle: The negatively charged nucleotide backbones form complexes with cationic polymers, such as diethyl- ibidi Solutions: aminoethyl (DEAE)-dextran . The complex is then taken up by the cells, mostly via endocytosis . If no The ibidi µ-Slides, µ-Dishes, and µ-Plates are lysosomal degradation occurs, the nucleotide can es- ideally suited for cationic polymer transfection cape from the endosome into the cytosol of the host using various cell numbers . cells, subsequently resulting in expression .

5 Viral Transduction

The term “transduction” is used to describe a virus-mediated transfer of nucleic acids into cells . In contrast to transfection of cells with foreign DNA or RNA, no transfection reagent is needed here . The , itself, also called virion, is able to infect cells and transport the DNA directly into the nucleus, independent of further actions . After the release of the DNA into the nucleus, the protein of interest is produced using the cells’ own machineries .

Adenoviral Transduction Virus Particle

Applications: Adenoviral Transduction Adenoviral vectors have proven to be a very successful CAR transduction tool in many eukaryotic cell types, such as human and rodent cells . Besides dividing cell lines, Endosome this method gives access to difficult-to-transfect cells, such as primary cells . Adenovirus-mediated

transduction is always transient, meaning that no Protein nucleotide integration into the host occurs .

Transduction efficiencies of up to 100% can easily be RELEASE OF DNA TRANSLATION achieved . Importantly, biosafety level S2 is needed mRNA for adenoviral transduction .

Principle:

Adenoviruses are a class of double stranded DNA that efficiently deliver nucleotides directly into target cells . After initial binding to the Coxsackievirus and adenovirus receptor (CAR), which is expressed ibidi Solutions: on the cell membrane, the virus enters the host cell via endocytosis . Following endosomal escape, the With the rAV-LifeAct viral genome is transported into the nucleus, where Adenoviral Vectors, it is expressed by the replication machineries of the ibidi provides a tool for host cell . In contrast to other viruses, the adenoviral visualizing F-actin in DNA remains episomal, i e. ,. it is not integrated into difficult-to-access cells the host genome . Nowadays, replication-deficient (e g. ,. endothelial cells) adenoviruses are widely used for transduction and gene without interfering with therapy, due to their high efficiency and low pathogenicity . cytoskeletal dynamics . The ibidi µ-Slides, µ-Dishes, and µ-Plates are Lee C.S., et al. Adenovirus-mediated : Potential ideally suited for transduction using various cell applications for gene and cell-based therapies in the new era of . Genes Dis, 2017, 10.1016/J. numbers and volumes . GENDIS.2017.04.001

Additional Material:

• AN 28: Adenoviral Transduction of Human Cells (PDF)

6 Lentiviral Transduction

ssRNA RELEASE OF RNA

REVERSE Protein Applications: TRANSCRIPTION

dsDNA Recombinant lentiviral vectors are powerful tools TRANSLATION mRNA Lentiviral for gene transfer with some advantages over other Transduction delivery vectors: Besides cells that undergo , Virus Particle they also have the ability to transduce non-dividing STABLE cells . Further, enable stable gene INTEGRATION transfer in vitro and in vivo, as they integrate into the host cell genome and offer the possibility of positive cell selection . They have a broad host cell range that also includes cell types such as primary neurons, lymphocytes, and macrophages . Moreover, ibidi Solutions: lentiviral vectors have also proven to be effective in transducing brain, liver, muscle, and retina in vivo The ibidi LifeAct Lenti- without toxicity or immune responses . Biosafety viral Vectors mediate level S2 is needed for lentiviral transduction . efficient transduction, inte- gration, and long-term expression of LifeAct into Principle: various cell types for reli- able F-actin visualization . Lentiviruses—a subclass of —have the ability to permanently integrate into the genome of The ibidi µ-Slides, µ-Dishes, and µ-Plates are the host cell . After the virus has entered the cell, the ideally suited for transduction using various cell viral RNA is transcribed by the reverse transcriptase numbers and volumes . to produce double-stranded DNA that enters the nucleus . Finally, the transgene is integrated into the host genome via the lentiviral integrase enzymes . When using lentiviral transduction, the user has to take into account effects caused by genomic integration . Additional Material:

Sakuma T., Barry M.A., Ikeda Y. Lentiviral vectors: basic to • AN 41: Lentiviral Transduction of Human translational. Biochem J, 2012, 10.1042/BJ20120146 Cells (PDF)

Method Comparison

Adenoviral transduction Lentiviral transduction Transfection

Viral genome material dsDNA RNA -

Transgene expression Transient Stable Transient or stable

Genome integration Episomal Integration into host Dependent on transfection genome method

Cell types Dividing and non-dividing Dividing and non-dividing Dividing and non-dividing cells cells cells, dependent on transfection method

Efficiency Up to 100% Up to 100% Variable transduction efficiency transduction efficiency transfection efficiency

Biosafety level 2 2 1

7 Physical Transfection

Microinjection

TRANSCRIPTION

Applications: mRNA

Microinjection allows for the efficient transfer of controlled nucleotide amounts into the nucleus of TRANSLATION Microinjection a specific target cell . It is a very precise, but time-

consuming and expensive transfection method with Protein a very low throughput only . Microinjection is mostly Plasmid used for special applications, such as single cell DNA manipulation or the generation of transgenic animals, e g. ,. by pronuclear injection in mice .

Principle:

With this physical method, the target cell is po- ibidi Solutions: sitioned under a microscope and being fixed by a pipette . The nucleotide solution is then directly ibidi provides specialized µ-Dishes with low injected into the cytoplasm and/or the nucleus using walls, which have been designed to give easy a fine glass capillary needle . This precise procedure access to the target cells for microinjection . The 35 mm, low 50 mm, low demands a rather expensive microinjection system μ-Dish and the μ-Dish enable and a lot of skill and practice . Once within the nucleus, exact positioning of micromanipulators for a the nucleotide can immediately integrate into the precise injection process . Further, their excellent endogenous DNA . Given these prerequisites, micro- optical quality allows for high-quality visualization injection is highly effective and offers the transfer of during microinjection . precisely controlled nucleotide amounts . However, as each single cell needs to be microinjected individually, this method is very time-consuming .

Electroporation TRANSCRIPTION

mRNA Applications:

Electroporation allows for the transient and stable TRANSLATION transfection of any cell type . This method is easy and

reliable, but it requires high cell numbers due to high Protein rates of cell death during the procedure . Therefore, Electroporation – electroporation is not suitable for sensitive and difficult- – + – + to-culture cell types, such as primary cells . Further, a + Plasmid special, expensive electroporation device is required . DNA

Principle:

During electroporation, a mixture of the cells and the nucleotide of interest is exposed to an intense electric field . This leads to transient cell membrane destabilization, making the cell membrane permeable to the nucleotides that are present in the surrounding solution . The cell membrane is transiently permeabilized by a short electric pulse, allowing the nucleotides, which are present in the surrounding solution, to enter the cytoplasm . After removing the electric field, the cell membrane stabilizes, enclosing the nucleotides in the cytoplasm, where they are expressed

8 Experimental Example

Actin Dynamics Under Flow

Several cell types in biofluidic vessels, such as endothelial cells and immune cells, are constantly exposed to shear stress in vivo . This mechanical stimulus has a great impact on the physiological behavior and adhesion properties of cells, and should be taken into account when performing respective studies . By combining the ibidi channel slides, µ-Slide I Luer or µ-Slide VI 0 4. , and the ibidi Pump System with ibidi’s LifeAct technology, the F-actin cytoskeleton can be visualized in living cells under shear stress conditions . The ibidi Pump System is ideal for the long-term application of physiological shear stress to a cell layer and enables the adjustment of different flow rates . The system is fully compatible with live cell imaging and high resolution fluorescence microscopy . Optionally, the fixation and immunofluorescence staining of the cells can be directly performed in the µ-Slide I Luer .

Experimental Setup:

• Device: ibidi Pump System • Slide: µ-Slide I 0 .4 Luer (ibiTreat) • Cells: LifeAct-expressing endothelial cells (HUVEC, P1), transduced with the LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2 • Reagents: LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2 Live cell imaging under flow: actin cytoskeleton visualization in • Shear stress parameters: 20 dyn/cm2 HUVEC after transduction with the LifeAct Adenoviral Vector rAV-LifeAct-TagGFP2 and cultivation under 20 dyn/cm2.

Click here to watch the movie on our website .

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